1. Field of the Invention
The invention relates to a new fluorimetric method for quantitative determination of human serum albumin (HSA) in biological liquids. The method is based on the observation that the intensity of the fluorescence of certain anionic cyanine dyes is intensified in the presence of human serum albumin and consequently provides an optical parameter for determination of human serum albumin.
2. Description of the Prior Art
Numerous methods for specific determination of albumin in biological liquids have been described in the literature: 1) The enzyme-linked immunosorbent assay (ELISA) is a very sensitive and selective immunological enzimatic method: Bergmeyer, Methods of Enzymatic Analysis, Third Edition, 1986, vol IX, p. 57. Detection of enzyme activity can be by either optical or other methods.
2) The radioimmuno assay (RIA) is a sensitive and selective method: J. Woo, M. Floyd, D. C. Cannon, B. Kahan, Radioimmunoassay for Urinary Albumin, Clin. Chem. 24, 1464-7 (1978).
3) Immunoelectrophesis is a sensitive and selective electrophoretic immunological method (C. B. Laurell, Quantitative Estimation of Protein by Electrophoresis in Agarose Gel Containing Antibodies, Anal. Biochem. 15, 45-52 (1966)).
4) Kinetic methods for determination are based on the intrinsic lipase activity of HSA. In these methods, the cleavage of a synthetic fatty acid ester which acts as an enzyme substrate is photometrically or fluorometrically observed as a function of time: A. R. Gurakar, O. S. Wolfbeis, A Sensitive Kinetic Assay of Serum Albumin Based on its Enzyme Like Hydrolytic Activity, Using a New Chromogenic and Fluorogenic Substrate, Clin. Chim. Acta 172, 35-46 (1988).
5) Finally, there are methods which are based on the spectral variations of dyes that bind to albumin: K. V. Waller, K. M. Ward, J. D. Mahan, D. K. Wismatt, Current Concepts in Proteinuria, Clin. Chem. 35 (5), 755-65 (1989). Among those, the best known is the CBB method: The dye Coomassie Brilliant Blue binds to HSA and changes its color from yellow to blue. This color change is caused by the change of the pK value of the dye following binding to HSA. As a result, the dye is converted into the blue conjugated base form: M. M. Bradford, A Rapid and Sensitive Method for the Quantitation of Microgram Quantities of Protein Utilizing the Principle of Protein-Dye binding, Anal. Biochem. 72, 248-54 (1976).